Peptides and compositions for prevention of cell adhesion and methods of using same

ABSTRACT

Compositions comprising an isolated peptide, which may for example optionally comprise a sequence comprising FNFDWY (SEQ ID NO: 5), or any cyclized version thereof, and methods of using same, including for treatment of or prevention of formation of microbial biofilms and against adhesion of a cell to a surface.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/142,358, filed on Jul. 12, 2011, which is the U.S. National Phase ofPCT/IB2009/007896, filed Dec. 28, 2009, which claims priority from U.S.Provisional Application No. 61/193,821, filed Dec. 29, 2008, which areincorporated herein by reference in entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 22, 2011, isnamed 09543201. txt and is 8,230 bytes in size.

FIELD OF THE INVENTION

The present invention relates to isolated peptides and their use inprevention of cell adhesion.

BACKGROUND OF THE INVENTION

Microorganisms can live and proliferate as individual cells swimmingfreely in the environment (as plankton), or they can grow as highlyorganized, multicellular communities encased in a self-producedpolymeric matrix in close association with surfaces and interfaces. Thelatter microbial lifestyle is referred to as biofilms. Biofilm formationrepresents an ancient, protected mode of growth that allows microbialsurvival in hostile environments and allows microorganisms to disperseand colonize new niches [Hall-Stoodley et al., Nat Rev Microbiol. (2004)2(2):95-108].

The composition of biofilms is complex and variable among differentmicrobial species and even within the same species under differentenvironmental conditions. Nonetheless, biofilm formation represents thenormal lifestyle of microorganism in the environment and all microbescan make biofilms. Previous studies revealed that bacterial biofilmformation progresses through multiple developmental stages differing inprotein profiles [Sauer et al., J Bacteriol. (2002) 184(4):1140-54],beginning with attachment to surface, followed by the immigration anddivision to form microcolonies and finally maturation involvingexpression of matrix polymers. Bacteria within each biofilm stagedisplay phenotypes and possess properties that are markedly differentfrom those of the same group growing planktonically [Sauer et al., JBacteriol. (2004) 186(21):7312-26].

Biofilms are a major cause of systemic infections (e.g. nosocomialinfections) in humans. In the body, biofilms can be associated withtissues (e.g., inner ears, teeth, gums, lungs, heart valves and theurogenital tract). An estimated 65% of bacterial infections in humansare biofilm in nature. Additionally, after forming biofilms,microorganisms tend to change their characteristics, sometimesdrastically, such that doses of antibiotics which normally kill theorganisms in suspended cultures are completely ineffective against thesame microorganisms when the organisms are in attached or conglomeratebiofilm form (U.S. Pat. No. 7,189,351).

One of the principal concerns with respect to products that areintroduced into the body (e.g., contact lenses, central venouscatheters, mechanical heart valves and pacemakers) or provide a pathwayinto the body is microbial infection and invariably biofilm formation.As these infections are difficult to treat with antibiotics, removal ofthe device is often necessitated, which is traumatic to the patient andincreases the medical cost. Accordingly, for such medical apparatuses,the art has long sought means and methods of rendering those medicalapparatuses and devices antimicrobial.

PCT Application No. WO 06/006172 discloses the use of anti-amyloidagents, such as aromatic compounds, for inhibiting formation ordisintegrating a pre existing biofilm. The application discloses thatcompounds preventing amyloid fibril formation in Alzheimers can actagainst fibril formation in biofilms, and concludes that amino acidshaving an aromatic arm are effective against biofilms. However, theanalysis was limited to full length sequences.

SUMMARY OF THE INVENTION

The present invention provides natural or synthetic peptides isolatedfrom animals, including mammals and non-mammals, that interfere withbiofilm formation at its initial stages, in a wide range ofmicroorganisms.

All peptides described herein show activity that is exclusively directedto the prevention of microbial substrate adhesion and the derivedbiofilm formation. It is devoid of the commonly observed lethalbactericidal activity, revealed by the antibiotic peptides and secondarymetabolites, which provides a strong selective pressure for rapidnatural selection by the intensive microbial “biotic potential.” On theother hand a wide range inhibition of microbial colonization antagonizesa fundamental mechanism of bacterial survival. Therefore an adaptivemodification of such mechanism has a low likelihood due to its vitality.

Sher et al. (Toxicon 45: 865-879, 2005) identified putative biologicallyactive proteins and polypeptides expressed by hydrae which could becomponents of its allomonal system, using a bioinformatics approach.Hydrae were shown to express orthologs of cnidarian phospholipase A2toxins and cytolysicns belonging to the actinoporin family, and toexpress proteins similar to clapid-like phospholipases, cysteine-richsecretory proctins (CRISP), prokineticin-like polypeptides and toxicdeoxyribonucleases.

The specific sequences responsible for cytotoxic activity in peptidesisolated from natural sources have not hitherto been identified.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

As used herein, the terms “comprising” and “including” or grammaticalvariants thereof are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofThis term encompasses the terms “consisting of” and “consistingessentially of.”

The phrase “consisting essentially of” or grammatical variants thereofwhen used herein are to be taken as specifying the stated features,integers, steps or components but do not preclude the addition of one ormore additional features, integers, steps, components or groups thereofbut only if the additional features, integers, steps, components orgroups thereof do not materially alter the basic and novelcharacteristics of the claimed composition, device or method.

The term “method” refers to manners, means, techniques and proceduresfor accomplishing a given task including, but not limited to, thosemanners, means, techniques and procedures either known to, or readilydeveloped from known manners, means, techniques and procedures bypractitioners of the chemical, biological and biophysical arts.

As used herein the term “about” refers to ±10%.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a bar graph showing inhibition of adherence of Pseudomonasaeruginosa by the synthetic peptide grZ28C [CSFSQNKSVHSFDYDWYNVSDQADLKNC(SEQ ID NO: 1)] at three different concentrations;

FIG. 2 is bar graph shows a growth test on Pseudomonas aeruginosa; and

FIG. 3 is a bar graph showing anti-adherence activity of peptides grZ35cyc and grZ28C based on the amino acid sequence of GPCR 137b onPseudomonas aeruginosa; and

FIG. 4 is a bar graph showing the effects of peptides grZ35 cyc andgrZ28C on P. aeruginosa growth.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of compositions comprising a peptide isolatedfrom an animal source, which has one or more effects relating toprevention of bacterial substrate adhesion and the derived biofilmformation, and optionally also prevention of cell-cell adhesion. Othereffects may also optionally be provided, additionally or alternatively.The peptide comprises at least the sequence FDYDWY (SEQ ID NO: 2). As anon-limiting example, the peptide may optionally and preferably comprisea sequence selected from the group consisting of FDYDWY (SEQ ID NO: 2),SFSQNKSVHSFDYDWYNVSDQADLKN (SEQ ID NO: 3) orCSFSQNKSVHSFDYDWYNVSDQADLKNC (SEQ ID NO: 1)

One of the major concerns in medicine is microbial biofilm formation. Inhumans, biofilms are a major concern when introducing products into thebody (e.g., contact lenses, central venous catheters, mechanical heartvalves and pacemakers).

Biofilms are also a problem in many industries including the food,pharmaceutical, paint, water, shipping and engineering industriescausing, amongst a wide range of negative effects, accelerated corrosionin industrial systems, oil souring and biofouling. For example,biofouling may be caused by the adhesion of organisms to any surface ina marine or freshwater environment, including cooling towers, waterpipes and filters in cooling or desalinization installations, irrigationand power stations, and membranes, such as those used in wastewater anddesalinization systems. Biofouling also occurs in aquaculture systems infish farms.

Furthermore the commercial shipping fleets of the world consumeapproximately 300 million tons of fuel annually. Without antifoulingmeasures, that fuel consumption would increase by as much as 40%,equivalent to an extra 120 million tons of fuel annually. The economiccost of this was estimated as about $7.5 billion in 2000; a more recentestimate is $30 billion.

Biofilms are very difficult to eliminate since microbes growing withinare highly organized and can withstand hostile environments, such ashigh temperatures and anti-microbial agents (e.g., antibiotics).

Marine and fresh water plants and organisms including soft bodied waterinvertebrates, fish and moss are surrounded by broad spectrum species ofmicrobial organisms. Since such plant and organisms lack specificimmunity, they produce several factors which can prevent microbialcolonization on their body surface.

The most “sensitive” organisms are invertebrates belong to the phylumcnidaria that include the sea anemones, corals, jellyfish, hydroids,medusae, and sea fans. Such soft bodied organism, which lack physicalprotection such as scales or shells, use proteins and secondarymetabolites to protect themselves from the microbial environmentsurrounding them.

It has been previously reported that marine organisms (e.g. sponges)produce secondary metabolites that exhibit antibacterial and antifungalactivities [Amade et al., supra]. Moreover, sea anemones (e.g., Actiniaequina) have been shown to produce toxic, pore forming peptides (i.e.,equinatoxins), which lyse and kill eukaryotic cells similarly to othersmall antimicrobial peptides [Anderluh et al., supra].

From these natural factors, peptides with high conservation sequenceswere isolated, and showed high activity in prevention of microbialadherence in its synthetic conformation. The conserved sequence is foundin several marine organisms, including various known species of seaanemone, several fish (including Danio rerio-zebra fish), and in mossPhyscomitrella patens subsp. Patens.

Based on bioinformatic analysis it is believed that the protein haschanged in upper organisms (including Homo sapiens) to FDYDWY (SEQ IDNO: 2), that can be found in proteins with size range from 128aa-400aa.In Homo sapiens this peptide is part of the G protein-coupled receptor137B (GENE ID: 7107 GPR137B) located at 269-274. Based onUniProtKB/Swiss-Prot entry O60478 the region, which starts at 259 andends at 292, is an extracellular region, which supports the theory thatthis peptide is the active part of the protein.

Bioinformatics analysis of the ancient sea organism Ciona intestinalisidentifieda 368 amino acid protein, similar to the G protein-coupledreceptor 137ba [ GeneBank Accesion number XP_(—)002125109]. The regionsimilar to the anti adhesive peptide is SPLRCSELSSFNFDWYNVSDQADLVN (SEQID NO: 4). Based on this information, and the fact that Cionaintestinalis is also exposed to a large diversity of microorganisms, thepresent inventors hypothesise that the peptide FNFDWY (SEQ ID NO: 5) isalso anti adhesive. The non-cyclized peptide sequenceSFSQNKSVHSFDYDWYNVSDQADLKN (SEQ ID NO: 3) which represent theextracellular region, residue 259-284, was synthesized with twoCysteines in C and N termini.

The cyclized peptide sequence SFSQNKSVHSFDYDWYNVSDQADLKNQLGDAGYV (SEQ IDNO: 6) which represents the extracellular region, residue 259-292, wassynthesized with two Cysteines in C and N termini and S-S bridged.

According to some embodiments, the peptide of the present inventioncomprises at least the sequence FDYDWY (SEQ ID NO: 2). For example, thepeptide may comprise at least one of FDYDWY (SEQ ID NO: 2), SFDYDWY (SEQID NO: 7), SFDYDWYN (SEQ ID NO: 8), HSFDYDWYN (SEQ ID NO: 9), HSFDYDWYNV(SEQ ID NO: 10), VHSFDYDWYNV (SEQ ID NO: 11), VHSFDYDWYNVS (SEQ ID NO:12), SVHSFDYDWYNVS (SEQ ID NO: 13), SVHSFDYDWYNVSD (SEQ ID NO: 14),KSVHSFDYDWYNVSD (SEQ ID NO: 15), KSVHSFDYDWYNVSDQ (SEQ ID NO: 16),NKSVHSFDYDWYNVSDQ (SEQ ID NO: 17), NKSVHSFDYDWYNVSDQA (SEQ ID NO: 18),QNKSVHSFDYDWYNVSDQA (SEQ ID NO: 19), QNKSVHSFDYDWYNVSDQAD (SEQ ID NO:20), SQNKSVHSFDYDWYNVSDQAD (SEQ ID NO: 21), SQNKSVHSFDYDWYNVSDQADL (SEQID NO: 22), FSQNKSVHSFDYDWYNVSDQADL (SEQ ID NO: 23),FSQNKSVHSFDYDWYNVSDQADLK (SEQ ID NO: 24), SFSQNKSVHSFDYDWYNVSDQADLK (SEQID NO: 25), SFSQNKSVHSFDYDWYNVSDQADLKN (SEQ ID NO: 3),CSFSQNKSVHSFDYDWYNVSDQADLKN (SEQ ID NO: 26) orCSFSQNKSVHSFDYDWYNVSDQADLKNC (SEQ ID NO: 1), or combinations thereof.

According to some embodiments, the peptide is cyclized.

According to some embodiments, the peptide is non-cyclized.

The peptide may be isolated from any animal. Preferably, the animal is avertebrate, such as, for example, a fish, an amphibian (including afrog, a toad, a newt or a salamander), a bird, a reptile (such as acrocodilee, a lizard, a snake, a turtle, a tortoise or a terrapin) or amammal (including a human).

The peptide is also present in the sea organism Ciona intestinalis,which belongs to the phylum Chordata. In this organism, the proteinshows similarity to the GPCR 137 b in upper vertebrae and since thisorganism is surrounded by microorganisms, the peptide that includes thesequence FNFDWY (SEQ ID NO: 5) is also part of the patent.

The peptide of the present invention may optionally comprise at leasttwo of the above sequences, connected by a linker of some type, suchthat the N-terminal of a first peptide sequence is connected to theC-terminal of the linker, and the C-terminal of a second peptidesequence is connected to the N-terminal of the linker.

As used herein, the term “linker” refers to any chemical bond ormolecule for connecting two peptides or for cyclizing a peptide asdescribed herein. The linker may also optionally comprise a polymer ofany suitable number of monomeric units. However, in any case preferablythe linker features an active group, and/or is derivatized to includesuch an active group, in at least two locations, so as to join two ormore peptides and/or to cyclize a peptide as described herein.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

According to one aspect of the present invention, there is provided acomposition comprising a peptide isolated from a human source, thepeptide comprising a sequence selected from the group consisting ofFDYDWY (SEQ ID NO: 2), SFSQNKSVHSFDYDWYNVSDQADLKN (SEQ ID NO: 3) orCSFSQNKSVHSFDYDWYNVSDQADLKNC (SEQ ID NO: 1).

According to an additional aspect of the present invention there isprovided a method of preventing adhesion of a single cell organism to asurface, the method comprising contacting the cell with a compositioncomprising a peptide isolated from a human source comprising a sequenceselected from the group consisting of FDYDWY (SEQ ID NO: 2),SFSQNKSVHSFDYDWYNVSDQADLKN (SEQ ID NO: 3) orCSFSQNKSVHSFDYDWYNVSDQADLKNC (SEQ ID NO: 1), thereby preventing adhesionof a cell to a surface.

According to some embodiments of the present invention, there ispreferably provided a domain which comprises at least one of the abovepeptides and which is effective against cell adhesion to a surface. Morepreferably, the domain is included as part of a protein. Optionally andmost preferably, the domain exhibits anti-adhesive behavior, for examplefor the prevention of formation and/or treatment of a biofilm, but doesnot exhibit cytotoxic behavior.

As used herein, the term “isolated” refers to a composition that hasbeen removed from its in-vivo location. Preferably the isolatedcompositions of the present invention are substantially free from othersubstances (e.g., other proteins that do not comprise anti-adhesiveeffects) that are present in their in-vivo location (i.e. purified orsemi-purified). The isolated peptides may optionally be synthetic orobtained from natural sources, including optionally by being expressedin-vivo using genetic engineering techniques.

According to some embodiments of the present invention, the compositionsof the present invention are devoid of cytotoxic or cytostatic activity,e.g. they are not bactericidal or bacteristatic.

According to some embodiments of the present invention, the compositionsof the present invention are resistant to lyophilization—e.g. theiractivities are preserved following freeze drying.

As used herein the phrase “single cell organism” refers to a unicellularorganism also termed a microorganism or a microbe. The single cellorganism of the present invention can be a eukaryotic single cellorganism (e.g., protozoa or fungi for example yeast) or a prokaryoticsingle cell organism (e.g., bacteria or archaea). The single cellorganisms of the present invention may be in any cellular environment,such as for example, in a biofilm, as isolated cells or as a cellsuspension.

As used herein the term “biofilm” refers to an extracellular matrix inwhich microorganisms are dispersed and/or form colonies. The biofilmtypically is made of polysaccharides and other macromolecules.

Exemplary bacterial cells, whose adhesion may be prevented according tothe method of the present invention, include gram positive bacteria andgram negative bacteria.

The term “Gram-positive bacteria” as used herein refers to bacteriacharacterized by having as part of their cell wall structurepeptidoglycan as well as polysaccharides and/or teichoic acids and arecharacterized by their blue-violet color reaction in the Gram-stainingprocedure. Representative Gram-positive bacteria include: Actinomycesspp., Bacillus anthracis, Bifidobacterium spp., Clostridium botulinum,Clostridium perfringens, Clostridium spp., Clostridium tetani,Corynebacterium diphtheriae, Corynebacterium jeikeium, Enterococcusfaecalis, Enterococcus faecium, Erysipelothrix rhusiopathiae,Eubacterium spp., Gardnerella vaginalis, Gemella morbillorum,Leuconostoc spp., Mycobacterium abscessus, Mycobacterium avium complex,Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacteriumhaemophilium, Mycobacterium kansasii, Mycobacterium leprae,Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacteriumsmegmatis, Mycobacterium terrae, Mycobacterium tuberculosis,Mycobacterium ulcerans, Nocardia spp., Peptococcus niger,Peptostreptococcus spp., Proprionibacterium spp., Sarcina lutea,Staphylococcus aureus, Staphylococcus auricularis, Staphylococcuscapitis, Staphylococcus cohnii, Staphylococcus epidermidis,Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcuslugdanensis, Staphylococcus saccharolyticus, Staphylococcussaprophyticus, Staphylococcus schleiferi, Staphylococcus similans,Staphylococcus warneri, Staphylococcus xylosus, Streptococcus agalactiae(group B streptococcus), Streptococcus anginosus, Streptococcus bovis,Streptococcus canis, Streptococcus equi, Streptococcus milleri,Streptococcus mitior, Streptococcus mutans, Streptococcus pneumoniae,Streptococcus pyogenes (group A streptococcus), Streptococcussalivarius, Streptococcus sanguis.

The term “Gram-negative bacteria” as used herein refer to bacteriacharacterized by the presence of a double membrane surrounding eachbacterial cell. Representative Gram-negative bacteria includeAcinetobacter calcoaceticus, Acinetobacter baumannii, Actinobacillusactinomycetemcomitans, Aeromonas hydrophila, Alcaligenes xylosoxidans,Bacteroides, Bacteroides fragilis, Bartonella bacilliformis, Bordetellaspp., Borrelia burgdorferi, Branhamella catarrhalis, Brucella spp.,Campylobacter spp., Chlamydia pneumoniae, Chlamydia psittaci, Chlamydiatrachomatis, Chromobacterium violaceum, Citrobacter spp., Eikenellacorrodens, Enterobacter aerogenes, Escherichia coli, Flavobacteriummeningosepticum, Fusobacterium spp., Haemophilus influenzae, Haemophilusspp., Helicobacter pylori, Klebsiella pneumoniae, Klebsiella spp.,Legionella spp., Leptospira spp., Moraxella catarrhalis, Morganellamorganii, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseriameningitidis, Pasteurella multocida, Plesiomonas shigelloides,Prevotella spp., Proteus spp., Providencia rettgeri, Pseudomonasaeruginosa, Pseudomonas spp., Rickettsia prowazekii, Rickettsiarickettsii, Rochalimaea spp., Salmonella spp., Salmonella typhi,Serratia marcescens, Shigella spp., Shigella sonnei, Treponema carateum,Treponema pallidum, Treponema pallidum endemicum, Treponema pertenue,Veillonella spp., Vibrio cholerae, Vibrio vulnificus, Yersiniaenterocolitica, Yersinia pestis.

The term “fungi” as used herein refers to the heterotrophic organismscharacterized by the presence of a chitinous cell wall, and in themajority of species, filamentous growth as multicellular hyphae.Representative fungi whose adhesion may be prevented according to themethod of the present invention include Candida albicans, Saccharomycescerevisiae, Candida glabrata, Candida parapsilosis and Candidadubliniensis.

As used herein the phrase “preventing adhesion” refers to reducing oreliminating cell attachment to a surface (e.g. by reducing the rate ofgrowth on a surface). Preferably, the compositions of the presentinvention prevent cell adhesion by as much as 10%, more preferably by20%, more preferably by 30%, more preferably by 40%, more preferably by50%, more preferably by 60%, more preferably by 70%, more preferably by80%, more preferably by 90% and most preferably by 100% as measured by acell adhesion assay. Exemplary cell adhesion assays are described hereinbelow and in the Examples section that follows. It will be appreciatedthat the compositions of the present invention may also be capable ofpreventing cell aggregation (i.e. cell aggregation not to a surface).

The present invention contemplates prevention of cellular adhesion to awide variety of surfaces including fabrics, fibers, foams, films,concretes, masonries, glass, metals, plastics, polymers, and like.

According to one embodiment, the surface is comprised in a device thatis susceptible to biofilm formation. Exemplary devices whose surfacesare contemplated by the present invention include, but are not limitedto, vessel hulls, automobile surfaces, air plane surfaces, membranes,filters, and industrial equipment.

The surface may also be comprised in medical devices, instruments, andimplants. Examples of such medical devices, instruments, and implantsinclude any object that is capable of being implanted temporarily orpermanently into a mammalian organism, such as a human. Representativemedical devices, instruments, and implants that may be used according tothe present invention include, for example, central venous catheters,urinary catheters, endotracheal tubes, mechanical heart valves,pacemakers, vascular grafts, stents and prosthetic joints. Methods ofpreventing cell attachment to medical devices and further examplesthereof are described herein below.

As mentioned, the method of the present invention is effected bycontacting the cell with a composition from an organism capable ofpreventing adhesion of the cell to a surface.

As used herein the term “contacting” refers to the positioning of thecompositions of the present invention such that they are in direct orindirect contact with the adhesive cells in such a way that the activeagent comprised within is able to prevent adhesion of cells thereto.Thus, the present invention contemplates both applying the compositionsof the present invention to a desirable surface and/or directly to theadhesive cells.

Contacting the compositions with a surface can be effected using anymethod known in the art including spraying, spreading, wetting,immersing, dipping, painting, ultrasonic welding, welding, bonding oradhering. The compositions of the present invention may be attached asmonolayers or multiple layers.

According to other embodiments of the present invention, the abovepeptides may optionally be altered so as to form non-peptide analogs,including but not limited to replacing one or more bonds with lesslabile bonds, cyclization (described in greater detail below) and thelike. Additionally or alternatively, a peptide may optionally beconverted to a small molecule through computer modeling, as describedfor example in PCT Application No. WO/2007/147098, hereby incorporatedby reference as if fully set forth herein.

A “peptidomimetic organic moiety” can optionally be substituted foramino acid residues in a peptide according to the present invention bothas conservative and as non-conservative substitutions. These moietiesare also termed “non-natural amino acids” and may optionally replaceamino acid residues, amino acids or act as spacer groups within thepeptides in lieu of deleted amino acids. The peptidomimetic organicmoieties optionally and preferably have steric, electronic orconfigurational properties similar to the replaced amino acid and suchpeptidomimetics are used to replace amino acids in the essentialpositions, and are considered conservative substitutions. However suchsimilarities are not necessarily required. The only restriction on theuse of peptidomimetics is that the composition at least substantiallyretains its physiological activity as compared to the native peptideaccording to the present invention.

Peptidomimetics may optionally be used to inhibit degradation of thepeptides by enzymatic or other degradative processes. Thepeptidomimetics can optionally and preferably be produced by organicsynthetic techniques. Non-limiting examples of suitable peptidomimeticsinclude D amino acids of the corresponding L amino acids, tetrazol(Zabrocki et al., J. Am. Chem. Soc. 110:5875 5880 (1988)); isosteres ofamide bonds (Jones et al., Tetrahedron Lett. 29: 3853 3856 (1988)); LL 3amino 2 propenidone 6 carboxylic acid (LL Acp) (Kemp et al., J. Org.Chem. 50:5834 5838 (1985)). Similar analogs are shown in Kemp et al.,Tetrahedron Lett. 29:5081 5082 (1988) as well as Kemp et al.,Tetrahedron Lett. 29:5057 5060 (1988), Kemp et al., Tetrahedron Lett.29:4935 4938 (1988) and Kemp et al., J. Org. Chem. 54:109 115 (1987).Other suitable but exemplary peptidomimetics are shown in Nagai andSato, Tetrahedron Lett. 26:647 650 (1985); Di Maio et al., J. Chem. Soc.Perkin Trans., 1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317(1989); Olson et al., J. Am. Chem. Soc. 112:323 333 (1990); Garvey etal., J. Org. Chem. 56:436 (1990). Further suitable exemplarypeptidomimetics include hydroxy 1,2,3,4 tetrahydroisoquinoline 3carboxylate (Miyake et al., J. Takeda Res. Labs 43:53 76 (1989));1,2,3,4 tetrahydro-isoquinoline 3 carboxylate (Kazmierski et al., J. Am.Chem. Soc. 133:2275 2283 (1991)); histidine isoquinolone carboxylic acid(HIC) (Zechel et al., Int. J. Pep. Protein Res. 43 (1991)); (2S,3S)methyl phenylalanine, (2S,3R) methyl phenylalanine, (2R,3S) methylphenylalanine and (2R,3R) methyl phenylalanine (Kazmierski and Hruby,Tetrahedron Left. (1991).

Exemplary, illustrative but non-limiting non-natural amino acids includebeta-amino acids (beta3 and beta2), homo-amino acids, cyclic aminoacids, aromatic amino acids, Pro and Pyr derivatives, 3-substitutedAlanine derivatives, Glycine derivatives, ring-substituted Phe and TyrDerivatives, linear core amino acids or diamino acids. They areavailable from a variety of suppliers, such as Sigma-Aldrich (USA) forexample.

In the present invention any part of a peptide may optionally bechemically modified, i.e. changed by addition of functional groups. Themodification may optionally be performed during synthesis of themolecule if a chemical synthetic process is followed, for example byadding a chemically modified amino acid. However, chemical modificationof an amino acid when it is already present in the molecule (“in situ”modification) is also possible.

The amino acid of any of the sequence regions of the molecule canoptionally be modified according to any one of the following exemplarytypes of modification (in the peptide conceptually viewed as “chemicallymodified”). Non-limiting exemplary types of modification includecarboxymethylation, acylation, phosphorylation, glycosylation or fattyacylation. Ether bonds can optionally be used to join the serine orthreonine hydroxyl to the hydroxyl of a sugar. Amide bonds canoptionally be used to join the glutamate or aspartate carboxyl groups toan amino group on a sugar (Garg and Jeanloz, Advances in CarbohydrateChemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang.Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds canalso optionally be formed between amino acids and carbohydrates. Fattyacid acyl derivatives can optionally be made, for example, by acylationof a free amino group (e.g., lysine) (Toth et al., Peptides: Chemistry,Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden,1078-1079 (1990)).

As used herein the term “chemical modification,” when referring to apeptide according to the present invention, refers to a peptide where atleast one of its amino acid residues is modified either by naturalprocesses, such as processing or other post-translational modifications,or by chemical modification techniques which are well known in the art.Examples of the numerous known modifications typically include, but arenot limited to: acetylation, acylation, amidation, ADP-ribosylation,glycosylation, GPI anchor formation, covalent attachment of a lipid orlipid derivative, methylation, myristylation, pegylation, prenylation,phosphorylation, ubiquitination, or any similar process.

As mentioned, medical devices and implants are commonly infected withopportunistic bacteria and other infectious microorganisms (e.g., fungi)in some cases necessitating the removal of implantable devices. Suchinfections can also result in illness, long hospital stays, or evendeath. The prevention of biofilm formation and infection of medicaldevices is therefore highly desirous.

Thus, the present invention also contemplates medical devices in whichthe above-described compositions are attached thereto.

As used herein the term “medical device” refers to any implant,instrument, apparatus, implement, machine, device or any other similaror related object (including any component or accessory), which isintended for use in the diagnosis, treatment, cure or prevention ofdisease or other conditions. Such medical device is intended for use inman or other animals and is anticipated to affect the structure or anyfunction of the body. Such medical device does not achieve its primaryintended purposes through chemical action and is not dependent uponbeing metabolized for the achievement of its primary intended purposes.

As used herein the term “implant” refers to any object intended forplacement in a human body that is not a living tissue. The implant maybe temporary or permanent. An implant can be an article comprisingartificial components, such as catheters or pacemakers. Implants canalso include naturally derived objects that have been processed so thattheir living tissues have been devitalized. As an example, bone graftsthat have been processed so that their living cells are removed(acellularized), but so that their shape is retained to serve as atemplate for ingrowth of bone from a host. As another example, naturallyoccurring coral can be processed to yield hydroxyapatite preparationsthat can be applied to the body for certain orthopedic and dentaltherapies.

The present invention therefore envisions coating medical devices withthe compositions of the present invention to prevent cell adherencethereto so as to reduce/eliminate any possible cell aggregation andbiofilm formation known to occur following implantation. Device-relatedinfections usually result from the introduction of microorganisms,primarily bacteria, during the device insertion or implantationprocedure, or from attachment of blood-borne organisms to the newlyinserted device and their subsequent propagation on its surface. Coatingthe medical device with the compositions of the present invention willtherefore inhibit biofilm formation of one or more microbial species,will prevent medical device related infections, and consequently willreduce the need of antibiotic treatment or removal of the medical devicefrom the subject.

Medical devices that may be coated according to the teachings of thepresent invention include, but not limiting to, artificial bloodvessels, catheters and other devices for the removal or delivery offluids to patients, artificial hearts, artificial kidneys, orthopedicpins, prosthetic joints, plates and implants; catheters and other tubes(including urological and biliary tubes, endotracheal tubes,peripherably insertable central venous catheters, dialysis catheters,long term tunneled central venous catheters, peripheral venouscatheters, short term central venous catheters, arterial catheters,pulmonary catheters, Swan-Ganz catheters, urinary catheters, peritonealcatheters), urinary devices (including long term urinary devices, tissuebonding urinary devices, artificial urinary sphincters, urinarydilators), shunts (including ventricular or arterio-venous shunts);prostheses (including breast implants, penile prostheses, vasculargrafting prostheses, aneurysm repair devices, mechanical heart valves,artificial joints, artificial larynxes, otological implants),anastomotic devices, vascular catheter ports, vascular stents, clamps,embolic devices, wound drain tubes, ocular lenses, dental implants,hydrocephalus shunts, pacemakers and implantable defibrillators,needleless connectors, voice prostheses and the like.

Another possible application of the compositions of the presentinvention is the coating of surfaces found in the medical and dentalenvironment. Such surfaces include the inner and outer aspects ofvarious instruments and devices, whether disposable or intended forrepeated uses. Such surfaces include the entire spectrum of articlesadapted for medical use, including without limitation, scalpels,needles, scissors and other devices used in invasive surgical,therapeutic or diagnostic procedures; blood filters. Other examples willbe readily apparent to practitioners in these arts.

Surfaces found in the medical environment also include the inner andouter aspects of pieces of medical equipment, medical gear worn orcarried by personnel in the health care setting. Such surfaces caninclude surfaces intended as biological barriers to infectious organismsin medical settings, such as gloves, aprons and faceshields. Commonlyused materials for biological barriers are thermoplastic or polymericmaterials such as polyethylene, dacron, nylon, polyesters,polytetrafluoroethylene, polyurethane, latex, silicone and vinyl. Othersurfaces can include counter tops and fixtures in areas used for medicalprocedures or for preparing medical apparatus, tubes and canisters usedin respiratory treatments, including the administration of oxygen, ofsolubilized drugs in nebulizers and of anesthetic agents. Other suchsurfaces can include handles and cables for medical or dental equipmentnot intended to be sterile. Additionally, such surfaces can includethose non-sterile external surfaces of tubes and other apparatus foundin areas where blood or body fluids or other hazardous biomaterials arecommonly encountered.

The compositions of the present invention can be used on the surface ofor within these medical devices to provide long term protection againstmicroorganism colonization and reduce the incidence of device-relatedinfections. These compositions can also be incorporated in combinationwith an anti-microbial agent (e.g., antibiotic agent) into coatings formedical devices. Such a combination will sufficiently kill or inhibitthe initial colonizing bacteria and prevent device-related infections aslong as the substance is presented in an inhibitory concentration at thedevice-microbe interface.

The compositions of the present invention can be directly incorporatedinto the polymeric matrix of the medical device at the polymer synthesisstage or at the device manufacture stage. The compositions can also becovalently attached to the medical device polymer. These and many othermethods of coating medical devices are evident to one of ordinary skillin the art.

Additional surfaces that can be treated according to the teachings ofthe present invention include the inner and outer aspects of thosearticles involved in water purification, water storage and waterdelivery, and those articles involved in food processing. Thus thepresent invention envisions coating a solid surface of a food orbeverage container to extend the shelf life of its contents.

Surfaces related to health can also include the inner and outer aspectsof those household articles involved in providing for nutrition,sanitation or disease prevention. Thus, the compositions of the presentinvention can be used for removal of disease-causing microorganisms fromexternal surfaces. These can include, for example food processingequipment for home use, materials for infant care, tampons, soap,detergents, health and skincare products, household cleaners and toiletbowls.

The surface may be also be laboratory articles including, but notlimited to, microscopic slide, a culturing hood, a Petri dish or anyother suitable type of tissue culture vessel or container known in theart.

The inventors of this application also envision the use of thecompositions of the present invention as anti-fouling agents.

As used herein the term “anti-fouling agents” refers to the compoundsused to protect underwater surfaces from attaching single cellorganisms. These single cell organisms include microorganism such asbacteria and fungi.

These underwater surfaces include any water immersed surface, includingships'/boats's hulls (i.e., the body or frame of a ship or boat),submergence vehicles, navigational aids, screens, nets, constructions,floating or emplaced offshore platforms (e.g., docks), buoys, signalingequipment and articles which come into contact with sea water or saltywater. Other underwater surfaces include structures exposed to sea waterincluding pilings, marine markers, undersea conveyances like cabling andpipes, fishing nets, bulkheads, cooling towers, and any device orstructure that operates submerged.

The compositions of the present invention can be incorporated intomarine coatings to limit undesirable marine fouling. Thus, theanti-fouling agents of the present invention can be formulated so as notto contain toxic materials (such as heavy metals), and still retaintheir efficacy. The anti-fouling paint of the present invention mayfurther contain binders(s), pigment(s), solvent(s) and additive(s).

Examples of solvents that may be used include aromatic hydrocarbons suchas xylene and toluene; aliphatic hydrocarbons such as hexane andheptane, esters such as ethyl acetate and butyl acetate; amides such asN-methylpyrrolidone and N,N-dimethylformamide; alcohols such asisopropyl alcohol and butyl alcohol; ethers such as dioxane, THF anddiethyl ether; and ketones such as methyl ethyl ketone, methyl isobutylketone and methyl isoamyl ketone. The solvents may be used alone or incombination thereof.

Examples of binders that may be used include alkyd resin, acrylic orvinyl emulsions, polyurethane resins, epoxy resins, silicone basedresins, acrylic resins, inorganic silicate based resins, vinyl resins,particularly a vinyl chloride/vinyl acetate copolymer, and rosin.

Examples of pigments that may be used include titanium dioxide, cuprousoxide, iron oxide, talc, aluminium flakes, mica flakes, ferric oxide,cuprous thiocyanate, zinc oxide, cupric acetate meta-arsenate, zincchromate, zinc dimethyl dithiocarbamate, zinc ethylenebis(dithiocarbamate) and zinc diethyl dithiocarbamate.

Examples of additives that may be incorporated into the coatingcomposition include dehumidifiers, wetting/dispersing agents,anti-settling agents, anti-skinning agents, drying/curing agents,anti-marring agents and additives ordinarily employed in coatingcompositions as stabilizers and anti-foaming agents. Additionally, anyantibiotic which is relatively insoluble in seawater can be used with ananti-fouling marine paint.

Methods of preparing marine anti-fouling paints are explained in detailin U.S. Pat. Nos. 4,678,512; 4,286,988; 4,675,051; 4,865,909; and5,143,545.

The compositions of the present invention may also be used for providingantibacterial properties in cosmetics, to prevent spoiling of theproduct.

The compositions may further be used to provide an antibacterial effectto the mouth, teeth and gums, such as by incorporation in a toothpaste,mouthwash, or chewing gum. Taken together the present teachings portraya wide range of novel anti-adhesive agents isolated from organisms suchas aquatic organisms and moss. The broad spectrum of the anti adhesioneffects of these agents (e.g. inhibiting adhesion of gram positive andgram negative bacteria) together with their ability to effect theinitial, vulnerable stages of microbial biofilm formation, makes theseagents prime candidates as anti-biofilm agents. Moreover, theanti-adhesive agents described herein are clonable enablingmodifications and mass production thereof. In addition their stability(i.e. resistance to environmental conditions) makes these agentssuitable for a diverse array of applications.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology,” JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning,” John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA,” Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series,” Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook,” Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology,”W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., Eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications,”Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

EXAMPLES

Reference is now made to the following examples, which together with theabove description, illustrate the invention in a non limiting fashion.

Example 1 Prevention of Bacterial Attachment by grZ-28C

The synthetic peptide grZ28C [CSFSQNKSVHSFDYDWYNVSDQADLKNC (SEQ ID NO:1)] gave approximately 50% prevention of Pseudomonas aeruginosaattachment at three concentrations: 50, 5 and 0.5 μg/ml (FIG. 1). Theactivity was similar to that of the AbacZ17C, peptide based on theanemone cytotoxin active region. Abac10C, which was used as a negativecontrol peptide, was synthesized based on the N-terminal sequence ofAbac17C, without the active residue [CMFSVPFDYC (SEQ ID NO: 27)].

FIG. 2 demonstrates that no growth effect occurs in the presence of thetest peptides.

FIG. 3 shows anti adherence activity with peptides grZ35 cyc and grZ28C,based on the amino acid sequence of GPCR 137b on P. aeruginosa. Forpeptide grZ35 cyc, the peptide sequenceSFSQNKSVHSFDYDWYNVSDQADLKNQLGDAGYV (SEQ ID NO: 6) which represents theextracellular region, residue 259-292, was synthesized with twoCysteines in the C and N termini and S-S bridged. For peptide grZ28C,the peptide sequence SFSQNKSVHSFDYDWYNVSDQADLKN (SEQ ID NO: 3) whichrepresents the extracellular region, residue 259-284, was synthesizedwith two Cysteines in C and N termini.

FIG. 4 shows the effects of peptides grZ35 cyc and grZ28C on P.aeruginosa growth, indicating that growth of the bacteria was notinhibited. This result is important as peptides of the present inventiondesirably show little or no growth inhibition of bacteria.

While the invention has been described with respect to a limited numberof embodiments, it will be appreciated that many variations,modifications and other applications of the invention may be made.

What is claimed is:
 1. A cyclized peptide consisting of a length of upto 26 amino acids and comprising FNFDWY (SEQ ID NO: 5), wherein thepeptide is cyclized at the C and N termini.
 2. A composition comprisinga cyclized peptide consisting of a length of up to 26 amino acids andcomprising FNFDWY (SEQ ID NO: 5), wherein the peptide is cyclized at theC and N termini.
 3. A method of inhibiting adhesion of a single cellorganism to a surface, the method comprising contacting the cell with acomposition comprising a cyclized peptide consisting of a length of upto 26 amino acids and comprising FNFDWY (SEQ ID NO: 5),wherein thepeptide is cyclized at the C and N termini.
 4. The composition of claim2, wherein the composition is lyophilized.
 5. The method of claim 3,further comprising said single cell organism in a biofilm.
 6. The methodof claim 3, wherein said single cell organism is selected from the groupconsisting of a bacteria, a fungus, a protozoa and an archaea.
 7. Themethod of claim 6, wherein said fungus is a yeast.
 8. The method ofclaim 3, wherein said surface is selected from the group consisting of afiber, a foam, a film, a concrete, a masonry, a glass, a metal and aplastic.
 9. A method of treating water to inhibit or reduce biofilmformation or fouling of a filter, comprising treating the water with thepeptide of claim 1 or the composition of claim
 2. 10. A method ofinhibiting or reducing biofilm formation in a fluid medium, comprisingtreating the fluid medium with the peptide of claim 1 or the compositionof claim
 2. 11. The method of claim 9, further comprising applying thetreated water or fluid medium to a reverse osmosis filter.
 12. Thepeptide of claim 1, wherein the peptide is synthetic.
 13. Thecomposition of claim 2, wherein the peptide is synthetic.
 14. The methodof claim 3, wherein the peptide is synthetic.
 15. The method of claim 9,wherein the peptide is synthetic.
 16. The method of claim 10, whereinthe peptide is synthetic.
 17. The method of claim 3, wherein thecomposition is attached to the surface.
 18. The method of claim 17,wherein the composition is attached to the surface as a monolayer or inmultiple layers.
 19. The method of claim 17, further comprising thesurface in a medical device.
 20. The method of claim 19, wherein themedical device is an extracorporeal device.
 21. The method of claim 19,wherein the medical device is an intracorporeal device.
 22. The methodof claim 3, further comprising said surface in a medical device.
 23. Themethod of claim 22, wherein the medical device is an extracorporealdevice.
 24. The method of claim 22, wherein the medical device is anintracorporeal device.